High elasticity hyaluronan compositions and methods of use thereof

ABSTRACT

The present invention provides methods for alleviating pain and discomfort associated with a dry eye condition; methods for alleviating pain and discomfort while minimizing at least one skin imperfection; and methods for alleviating pain and discomfort while facilitating wound healing. The methods involve administering to a subject in need thereof a composition comprising hyaluronan with high elasticity.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/460,557, filed on Jul. 2, 2019; which is a continuation of U.S.application Ser. No. 15/271,434, filed on Sep. 21, 2016, now U.S. Pat.No. 10,383,889, issued on Aug. 20, 2019; which claims the benefit ofU.S. Provisional Application Ser. No. 62/232,364, filed on Sep. 24,2015. The entire contents of each of the foregoing applications arehereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Hyaluronan or hyaluronic acid (HA) is a high average molecular weightlinear polysaccharide which is distributed widely throughout connective,epithelial, and neural tissues. HA is found primarily in theextracellular matrix and pericellular matrix, but has also been shown tooccur intracellularly. The biological functions of HA includemaintenance of the elastoviscosity of liquid connective tissues such assynovial fluid in the joints and the vitreous of the eye, control oftissue hydration and water transport, supramolecular assembly ofproteoglycans in the extracellular matrix, and numerousreceptor-mediated roles in cell detachment, mitosis, migration and tumordevelopment.

Some of the known uses of HA include treatment of dry eye conditions, inskin care as dermal fillers and to promote wound healing. Often, dry eyeconditions, skin care/dermatological procedures and wound healing areassociated with pain and discomfort that typically require a separateadministration of an analgesic medication. The HA formulations currentlyused to treat the above conditions are not effective in treating topicalpain. Accordingly, there is a need in the art for methods to treat dryeye conditions effectively, to minimize skin imperfections withinjectable augmentation devices, and to promote wound healing whilealleviating the pain associated with these conditions.

SUMMARY OF THE INVENTION

The present inventors have discovered that compositions comprising highconcentrations of HA, e.g., compositions having HA concentrations ofabout 30 mg/mL (about 3% weight/volume) or greater can be usedeffectively to alleviate pain and discomfort associated with dry eyeconditions, dermatological procedures, and healing wounds. Withoutwishing to be bound by a specific theory, it is believed that theeffectiveness of the HA compositions of the invention comprising highconcentrations of HA for treating pain and discomfort is determined bytheir high elasticity, as is evidenced by the high value of the elasticmodulus G′. It is also believed, without wishing to be bound by aspecific theory, that the effectiveness of the HA compositions of theinvention is determined by a relatively high probability of interactionof HA molecules with pain transducing channels, such as TRPV1, therebyreducing nociceptor excitability.

Accordingly, the present invention provides a method for alleviatingpain and discomfort associated with a dry eye condition in a subject inneed thereof. The method comprises administering to an eye of thesubject a composition, e.g., a pharmaceutical composition, comprisinghyaluronan, wherein: the hyaluronan is present in the composition at aconcentration of greater than about 30 mg/mL, e.g., about 35 mg/mL ormore, about 40 mg/mL or more, about 45 mg/mL or more, about 50 mg/mL ormore, about 55 mg/mL or more, about 60 mg/mL or more, about 65 mg/mL ormore, about 70 mg/mL or more, about 75 mg/mL or more, about 80 mg/mL ormore, about 85 mg/mL or more, about 90 mg/mL or more, about 95 mg/mL ormore or about 100 mg/mL or more; the hyaluronan has an average molecularweight of between about 1 and about 2 million; and the hyaluronan is notcross-linked and/or is substantially free of chemical modifications,thereby alleviating the pain and discomfort in the subject.

In another aspect, the hyaluronan is present in the composition at aconcentration of about 40 mg/mL to about 60 mg/mL.

In some embodiment, the present invention also provides a method foralleviating pain and discomfort associated with a dry eye condition in asubject in need thereof, which comprises administering to an eye of thesubject a composition, e.g., a pharmaceutical composition, comprisinghyaluronan, wherein: the hyaluronan is present in the composition at aconcentration of at least about 40 mg/mL; the hyaluronan has an averagemolecular weight of between about 1 and about 2 million; and thehyaluronan is not cross-linked and/or is substantially free of chemicalmodifications, thereby alleviating the pain and discomfort in thesubject.

In some embodiments, the composition is substantially free of otherpharmaceutically active substances.

In certain aspects, the composition does not comprise a polyglycol.

In some embodiments, the composition further comprises a buffer, e.g.,phosphate buffered saline (PBS).

In some embodiments, the composition has an elasticity of at least about200 Pascal when measured at a frequency of 0.5 Hz; at least about 1,000Pascal when measured at a frequency of 0.5 Hz; at least about 2,000Pascal when measured at a frequency of 0.5 Hz; or at least about 4,000Pascal when measured at a frequency of 0.5 Hz.

In some embodiments, the composition is in the form of a gel, anointment, a liniment, a lotion or a cream.

In some aspects, the composition is administered to the ocular surface,such as under the eye lid, e.g., under the upper or lower eye lid, ofthe subject or at the cornea-eyelid interface.

In certain embodiments, the composition is administered to the subjectimmediately prior to rest or sleep.

In some aspects, the composition is administered without an injectioninto the eye of the subject.

In some aspects, the composition is administered using a container,e.g., a single dose container, such as a soft plastic bottle, a tube, anairless tube, an eye cup, a dropper or a cartridge.

In some embodiments, the dry eye condition is associated with one ormore symptoms selected from the group consisting of ocular dryness;decreased tear production, volume, and flow; abnormal tear composition;increased tear osmolarity; keratitis; conjunctival and corneal staining;redness; blurry vision; decreased tear film break-up time; increasedconjunctival redness; excess debris in tear film, ocular grittiness;ocular burning; foreign body sensation in the eye; excess tearing;photophobia; ocular stinging; refractive impairment; ocular sensitivity;and ocular irritation. In other embodiments, the dry eye condition isassociated with a condition selected from the group consisting of anautoimmune disorder; an ocular surgery; ingestion of a medication; dryenvironmental conditions; prolonged computer use; ocular fatigue;prolonged contact lens wear, corneal sensitivity; partial lid closure;surface irregularities; eye lid irregularities; and a conditionassociated with corneal nociceptive pain associated with corneal injuryor a condition associated with neuropathic pain. In one furtherembodiment, the dry eye condition is associated with an ocular surgery,and the ocular surgery is selected from the group consisting ofphotorefractive surgery, such as photorefractive keratectomy (PRK),cataract surgery, retinal detachment surgery, laser-assisted in situkeratomileusis (LASIK), and any corneal surgical procedure involvingdamage to corneal sensory nerves. In a further embodiment, the dry eyecondition is associated with a condition associated with neuropathicpain, e.g., a cataract or a retinal detachment, or surgery designed totreat cataract or retinal detachment.

In some aspects, the composition is administered daily for 3 days, 4days, 5 days, 6 days, 1 week, 2 weeks, 4 weeks or 10 weeks. In otheraspects, long term amelioration, e.g., 8 hours, 12 hours, 24 hours, 1day, 3 days, 5 days, 7 days, 14 days or 28 days, of the pain anddiscomfort is achieved in the subject.

In some embodiments, the subject is a mammal, e.g., a human.

In yet another aspect, the present invention also provides a method foralleviating pain and discomfort while minimizing at least one skinimperfection in a subject in need thereof. The method comprisesadministering to the subject a composition comprising hyaluronan,wherein: the hyaluronan is present in the composition at a concentrationof greater than about 30 mg/mL; the hyaluronan has an average molecularweight of between about 1 and about 2 million; the hyaluronan is notcross-linked and/or is substantially free of chemical modifications; andwherein the composition is substantially free of other pharmaceuticallyactive substances, thereby alleviating the pain and minimizing the atleast one skin imperfection.

In some embodiments, the hyaluronan is present at a concentration ofabout 40 mg/mL to about 60 mg/mL.

In a further aspect, the present invention also provides a method foralleviating pain and discomfort while minimizing at least one skinimperfection in a subject in need thereof, which comprises administeringto the subject a composition comprising hyaluronan, wherein: thehyaluronan is present in the composition at a concentration of at leastabout 40 mg/mL; the hyaluronan has an average molecular weight ofbetween about 1 and about 2 million; the hyaluronan is not cross-linkedand/or is substantially free of chemical modifications; and wherein thecomposition is substantially free of other pharmaceutically activesubstances, thereby alleviating the pain and minimizing the at least oneskin imperfection.

In some embodiments, the other pharmaceutically active substance is alocal anesthetic, e.g., lidocaine or bupivacaine.

In certain aspects, the composition also comprises a buffer, e.g.,phosphate buffered saline (PBS).

In some embodiments, the composition has an elasticity of at least about200 Pascal when measured at a frequency of 0.5 Hz; at least about 1,000Pascal when measured at a frequency of 0.5 Hz; at least about 2,000Pascal when measured at a frequency of 0.5 Hz; or at least about 4,000Pascal when measured at a frequency of 0.5 Hz.

In some aspects, the composition is sterile.

In certain embodiments, the composition is administered by an injectioninto the skin of the subject. In a further embodiment, the compositionis injected into the face of the subject. For example, the compositionis injected into a region selected from the group consisting ofnasolabial region, upper lip region, forehead, eye region and cheekregion.

In some aspects, the composition is administered by an injection using apre-filled syringe, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-mLpre-filled syringe. In a further aspect, the pre-filled syringe issterilized.

In another aspect, the present invention also provides a method foralleviating pain and discomfort while facilitating wound healing in asubject in need thereof. The method comprises administering to thesubject a composition, e.g., a pharmaceutical composition, comprisinghyaluronan, wherein: the hyaluronan is present in the composition at aconcentration of greater than about 30 mg/mL; the hyaluronan has anaverage molecular weight of between about 1 and about 2 million; and thehyaluronan is not cross-linked and/or is substantially free of chemicalmodifications, thereby alleviating the pain and facilitating the woundhealing.

In one further embodiment, the hyaluronan is present at a concentrationof about 40 mg/mL to about 60 mg/mL.

In a further aspect, the present invention also provides a method foralleviating pain and discomfort while facilitating wound healing in asubject in need thereof, which comprises administering to the subject acomposition, e.g., a pharmaceutical composition, comprising hyaluronan,wherein: the hyaluronan is present in the composition at a concentrationof at least about 40 mg/mL; the hyaluronan has an average molecularweight of between about 1 and about 2 million; and the hyaluronan is notcross-linked and/or is substantially free of chemical modifications,thereby alleviating the pain and facilitating the wound healing.

In some aspects, the composition is substantially free of otherpharmaceutically active substances, such as local anesthetics, e.g.,lidocaine or bupivacaine.

In some embodiments, the composition further comprises a buffer, e.g.,phosphate buffered saline (PBS).

In some aspects, the composition has an elasticity of at least about 200Pascal when measured at a frequency of 0.5 Hz; at least about 1,000Pascal when measured at a frequency of 0.5 Hz; at least about 2,000Pascal when measured at a frequency of 0.5 Hz; or at least about 4,000Pascal when measured at a frequency of 0.5 Hz.

In some embodiments, the composition is sterile.

In certain embodiments, the composition is administered topically, e.g.,on the surface of a wound or a scar on the skin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the pressure required to eject a 4% HAsolution through needles of different sizes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for alleviating pain anddiscomfort associated with a dry eye condition; skin care/dermatologicaltreatments and wound healing. The presently claimed methods compriseadministering HA compositions comprising high concentrations ofhyaluronan (HA), e.g., greater than about 30 mg/mL or more. Suchcompositions were determined to have high elasticity, e.g., high elasticmodulus G′, when measured at frequencies of 0.1-10 Hz. HA compositionscharacterized by high elasticity, e.g., compositions comprising highconcentrations of HA, are surprisingly effective at alleviating pain anddiscomfort resulting from a dry eye condition; a cosmetic treatment; orwound healing. Without wishing to be bound by a specific theory, it isbelieved that the effectiveness of the HA compositions of the inventioncomprising high concentrations of HA at treating pain and discomfort isdetermined by their high elasticity, as is evidenced by the high valueof the elastic modulus G′. It is also believed, without wishing to bebound by a specific theory, that the effectiveness of the HAcompositions of the invention is determined by a relatively highprobability of interaction of HA molecules with pain transducingchannels, such as TRPV1, thereby reducing nociceptor excitability. Theaverage molecular weight of HA used in the methods of the invention maybe 2 million or less, e.g., between about 1-2 million.

I. Hyaluronan Compositions for Use in the Methods of the Invention

The present invention provides methods that comprise administering to asubject in need thereof compositions comprising hyaluronan (HA). In someembodiments, the composition comprises hyaluronan, wherein thehyaluronan is present in the composition at a concentration of greaterthan about 30 mg/mL (or greater than about 3% weight/volume); thehyaluronan has an average molecular weight of between about 1 and about2 million; and the hyaluronan is not cross-linked and/or issubstantially free of chemical modifications.

For example, the hyaluronan concentration in the composition may beabout 30 mg/mL (or about 3% w/v), about 35 mg/mL (or about 3.5% w/v),about 40 mg/mL (or about 4% w/v), about 45 mg/mL (or about 4.5% w/v),about 50 mg/mL (or about 5% w/v), about 55 mg/mL (or about 5.5% w/v),about 60 mg/mL (or about 6% w/v), about 65 mg/mL (or about 6.5% w/v),about 70 mg/mL (or about 7% w/v), about 75 mg/mL (or about 7.5% w/v),about 80 mg/mL (or about 8% w/v), about 85 mg/mL (or about 8.5% w/v),about 90 mg/mL (or about 9% w/v), about 95 mg/mL (or about 9.5% w/v),about 100 mg/mL (or about 10% w/v), about 105 mg/mL (or about 10.5%w/v), about 110 mg/mL (or about 11% w/v) about 115 mg/mL (or about 11.5%w/v), about 120 mg/mL (or about 12% w/v), about 125 mg/mL (or about12.5% w/v), about 130 mg/mL (or about 13% w/v), about 135 mg/mL (orabout 13.5% w/v), about 140 mg/mL (or about 14% w/v), about 145 mg/mL(or about 14.5% w/v), or about 150 mg/mL (or about 15% w/v). In aspecific embodiment, the HA is present in the composition at aconcentration of about 40 mg/mL (or about 4% w/v). In other specificembodiments, the HA is present in the composition of the invention atthe concentration of about 41 mg/mL (or about 4.1% w/v), about 42 mg/mL(or about 4.2% w/v), about 43 mg/mL (or about 4.3% w/v), about 44 mg/mL(or about 4.4% w/v), about 45 mg/mL (or about 4.5% w/v), about 46 mg/mL(or about 4.6% w/v), about 47 mg/mL (or about 4.7% w/v), about 48 mg/mL(or about 4.8% w/v), about 49 mg/mL (or about 4.9% w/v), about 50 mg/mL(or about 5.0% w/v), about 51 mg/mL (or about 5.1% w/v), about 52 mg/mL(or about 5.2% w/v), about 53 mg/mL (or about 5.3% w/v), about 54 mg/mL(or about 5.4% w/v), about 55 mg/mL (or about 5.5% w/v), about 56 mg/mL(or about 5.6% w/v), about 57 mg/mL (or about 5.7% w/v), about 58 mg/mL(or about 5.8% w/v), 59 mg/mL (or about 5.9% w/v) or about 60 mg/mL (orabout 6% w/v).

In some examples, the hyaluronan concentration in the composition may begreater than about 30 mg/mL (or about 3% w/v), e.g., greater than about31 mg/mL (or about 3.1%), greater than about 32 mg/mL (or about 3.2%),greater than about 33 mg/mL (or about 3.3%), greater than about 34 mg/mL(or about 3.4%), greater than about 35 mg/mL (or about 3.5%), greaterthan about 36 mg/mL (or about 3.6%), greater than about 37 mg/mL (orabout 3.7%), greater than about 38 mg/mL (or about 3.8%), or greaterthan about 39 mg/mL (or about 3.9%).

In some examples, the hyaluronan concentration in the composition mayhave an average molecular weight of between about 1 and about 2 million;e.g., between about 1.1 and about 2 million, between about 1.2 and about2 million, between about 1.3 and about 2 million, between about 1.4 andabout 2 million, between about 1.5 and about 2 million or between about1.6 and about 2 million.

In certain embodiments, the hyaluronan used in the compositions is notcross-linked and/or is free of chemical modifications. For example, thehyaluronan used in the compositions is free from amidation that may beformed by a reaction between the carboxyl group of HA and the aminegroup of a derivatizing agent as described, e.g., in EP Patent No.1095064 B1. The hyaluronan used in the compositions may also be freefrom chemical modifications and/or cross-links that may result from thereaction of hyaluronan with a carbodiimide, such as a monocarbodiimideor a biscarbodiimide, as described, for example, in U.S. Pat. No.8,323,617. In some cases, the hyaluronan used in the compositions mayalso be free from acrylates, e.g., methacrylates as described in U.S.Publication No. 2010/0048755; sulfates as described, e.g., in U.S.Publication No. 2013/0209531; and deuterium, as described, e.g., in U.S.Publication No. 2015/0148310.

In some embodiments, the HA compositions of the invention are free fromother pharmaceutically active substances. As used herein, a“pharmaceutically active substance” is a substance that is capable ofexerting a biological effect on a subject, e.g., a human or an animalsubject. The term “pharmaceutically active substance” also comprisessubstances that can modulate the biological effect of an HA compositionwhen the composition is administered to a subject, e.g., alleviate painand discomfort associated with a dry eye condition; a skincare/dermatological treatment, or a healing wound. In certainembodiments, the pharmaceutically active substance is a protein, e.g., abone morphogenic protein (BMP), such as rhGDF-5. In certain embodiments,the pharmaceutically active substance is a glycosaminoglycan (GAG) thatis different from HA, e.g., chondroitin. In some embodiments, thepharmaceutically active substance is hydroxypropyl methyl cellulose. Inother embodiments, the pharmaceutically active substance is a topicalanesthetic, such as a lidocaine or a bupivacaine. In some cases, thepharmaceutically active substance is a purinergic receptor agonist,e.g., a P2Y₂ receptor agonist.

In certain embodiments, the HA compositions of the invention are freefrom molecules capable of scavenging free radicals, such as a polyol,e.g., sorbitol, maltitol, xylitol or isomalt. In other embodiments, theHA compositions used in the methods of the invention are free frommolecules that diminish the elasticity of HA, for example, dextran orsucrose.

In some cases, the HA compositions used in the methods of the inventionare free from a polyglycol, e.g., polyethylene glycol.

An HA composition may consist essentially of HA present at aconcentration of greater than about 30 mg/mL (about 3% w/v), or about 40mg/mL (about 4% w/v) in a physiological buffer, e.g., a phosphate bufferor a bicarbonate buffer, and having the average molecular weight ofbetween about 1 million and about 2 million. For example, an HAcomposition used in the methods of the invention consists essentially ofHA present at a concentration of about 40 mg/mL (or about 4% w/v), andhaving the average molecular weight of between about 1 million and about2 million.

The hyaluronan in the compositions used in the methods of the inventionmay have an elasticity of at least 100 Pascal when measured at afrequency of 0.5 Hz, or an elasticity of at least 400 Pascal whenmeasured at a frequency of 0.5 Hz, or an elasticity of at least 1,000Pascal when measured at a frequency of 0.5 Hz, or an elasticity of atleast 2,000 Pascal when measured at a frequency of 0.5 Hz, or anelasticity of at least 4,000 Pascal when measured at a frequency of 0.5Hz, or an elasticity of between 400 and 5,000 Pascal when measured at afrequency of 0.5 Hz.

It should be appreciated that a variety of methods are available formeasuring the elasticity of a biopolymer such as hyaluronan. In oneembodiment, the elasticity of compositions comprising hyaluronan ismeasured as pressure (expressed in Pascal) at a specific frequency(expressed in Hertz). For instance, the frequencies that may be used toevaluate the elasticity of the hyaluronan compositions provided herein,may be measured at 0.5 Hz, 2.5 Hz, or 5.0 Hz.

It should further be appreciated that the elasticity may be expressed inany relevant frequency. Thus, for instance, in one embodiment, theelasticity is expressed based on a frequency of 2.5 Hz and a compositioncomprising hyaluronan with high elasticity is a composition with anelasticity of at least 200 Pa at a frequency of 2.5 Hz. Similarly, inone embodiment, the elasticity is expressed based on a frequency of 5.0Hz and a composition comprising hyaluronan with high elasticity is acomposition having an elasticity of at least 400 Pa at a frequency of5.0 Hz.

In one embodiment, a composition comprising hyaluronan for use in thepresently claimed methods has an elasticity of at least 100 Pascal whenmeasured at a frequency of 0.5 Hz. In some embodiments, the compositionhas an elasticity of at least 300 Pascal when measured at a frequency of2.5 Hz. In some embodiments, the composition has an elasticity of atleast 350 Pascal when measured at a frequency of 5.0 Hz.

In a further embodiment, a composition comprising hyaluronan has anelasticity of at least 400 Pascal when measured at a frequency of 0.5Hz. In some embodiments, the composition has an elasticity of at least750 Pascal when measured at a frequency of 2.5 Hz. In some embodiments,the composition has an elasticity of at least 900 Pascal when measuredat a frequency of 5.0 Hz.

In another embodiment, a composition comprising hyaluronan has anelasticity of at least 1000 Pascal when measured at a frequency of 0.5Hz. In some embodiments, the composition has an elasticity of at least1600 Pascal when measured at a frequency of 2.5 Hz. In some embodiments,the composition has an elasticity of at least 2000 Pascal when measuredat a frequency of 5.0 Hz.

In yet another embodiment, a composition comprising hyaluronan has anelasticity of at least 2600 Pascal when measured at a frequency of 0.5Hz. In some embodiments, the composition has an elasticity of at least4000 Pascal when measured at a frequency of 2.5 Hz. In some embodiments,the composition has an elasticity of at least 4500 Pascal when measuredat a frequency of 5.0 Hz.

In one embodiment, a composition comprising hyaluronan has an elasticityof at least 4000 Pascal when measured at a frequency of 0.5 Hz. In someembodiments, the composition has an elasticity of at least 5000 Pascalwhen measured at a frequency of 2.5 Hz. In some embodiments, thecomposition has an elasticity of at least 6000 Pascal when measured at afrequency of 5.0 Hz.

In some embodiments, the composition has an elasticity of between 100and 10,000 Pascal when measured at a frequency of 0.5 Hz. In someembodiments, the composition has an elasticity of between 400 and 5,000Pascal when measured at a frequency of 0.5 Hz. In some embodiments, thecomposition has an elasticity of between 1,000 and 2,000 Pascal whenmeasured at a frequency of 0.5 Hz.

In some embodiments, the composition has an elasticity of between 300and 10,000 Pascal when measured at a frequency of 2.5 Hz. In someembodiments, the composition has an elasticity of between 750 and 6,000Pascal when measured at a frequency of 2.5 Hz. In some embodiments, thecomposition has an elasticity of between 1,500 and 4,000 Pascal whenmeasured at a frequency of 2.5 Hz.

In some embodiments, the composition has an elasticity of between 300and 10,000 Pascal when measured at a frequency of 5.0 Hz. In someembodiments, the composition has an elasticity of between 900 and 7,000Pascal when measured at a frequency of 5.0 Hz. In some embodiments, thecomposition has an elasticity of between 2,000 and 5,000 Pascal whenmeasured at a frequency of 5.0 Hz.

In some embodiments, the elasticity may be measured by using a suitabledevice (e.g., a rheometer). For example, the elasticity may be measuredby using a Stresstech High Resolution Research Rheometer (ReologicaInstruments AB). Typically, the elasticity is determined at ambienttemperature and pressure; however, it should be appreciated thatelasticity may also be measured at non-ambient temperature and/orpressure. It should further be appreciated that a person of ordinaryskill in the art knows how to convert a magnitude of elasticitydetermined at various temperatures and pressures into a magnitude ofelasticity at ambient temperature and pressure.

High elasticity compositions of hyaluronan can be prepared by increasingthe concentration of hyaluronan in the composition. Thus, in one aspect,the presently claimed methods utilize compositions having highelasticity that comprise a high percentage of hyaluronan. For example,the compositions may comprise at least 3.0% of hyaluronan (weight byvolume), at least 3.5% of hyaluronan (weight by volume), at least 4.0%of hyaluronan (weight by volume), at least 4.5% of hyaluronan (weight byvolume), at least 5.0% of hyaluronan (weight by volume), at least 5.5%of hyaluronan (weight by volume), at least 6.0% of hyaluronan (weight byvolume), at least 6.5% of hyaluronan (weight by volume), at least 7.0%of hyaluronan (weight by volume), at least 7.5% of hyaluronan (weight byvolume), at least 8.0% of hyaluronan (weight by volume), at least 8.5%of hyaluronan (weight by volume), at least 8.9% of hyaluronan (weight byvolume), at least 9.0% of hyaluronan (weight by volume), at least 10.0%of hyaluronan (weight by volume), at least 11.0% of hyaluronan (weightby volume), at least 12.0% of hyaluronan (weight by volume), at least13.0% of hyaluronan (weight by volume), at least 14.0% of hyaluronan(weight by volume), or at least 15.0%, or more, of hyaluronan (weight byvolume).

Ranges intermediate to the recited values are also intended to beincluded in the compositions for use in the methods of this invention.For example, hyaluronan content in the compositions may be between about3% and about 15% (weight/volume), between about 3% and about 10%(weight/volume), about 3.5% and about 9% (weight/volume), about 4% andabout 8% (weight/volume), or about 5% and about 7% (weight/volume).

It should further be appreciated that the amount of hyaluronan in aparticular volume may also be expressed by alternative means (e.g.,gram/liter or mol/liter). A person of ordinary skill in the art wouldknow how to convert the various means of expressing the amount ofhyaluronan in a particular volume.

Compositions of hyaluronan with a high concentration of hyaluronan, evenwith an average molecular weight of about 1-2 million, are particularlyeffective in the treatment of pain, e.g., pain associated with a dry eyecondition, a cosmetic treatment or a healing wound. Thus, the hyaluronancomprised in the HA compositions described herein may have an theaverage molecular weight that falls within the range of between 1 and 2million and is also less than 2 million, less than 1.9 million, lessthan 1.8 million, less than 1.7 million, less than 1.6 million, lessthan 1.5 million, less than 1.4 million, less than 1.3 million, lessthan 1.2 million, less than 1.1 million, less than 1 million, less than0.9 million, less than 0.8 million, less than 0.7 million, less than 0.6million, or less than 0.5 million. In other cases, the hyaluronancomprised in the HA compositions described herein may have an theaverage molecular weight that falls within the range of between 1 and 2million and is also greater than 0.1 million, greater than 0.2 million,greater than 0.3 million, greater than 0.4 million, greater than 0.5million, greater than 0.6 million, greater than 0.7 million, greaterthan 0.8 million, greater than 0.9 million, greater than 1 million,greater than 1.1 million, greater than 1.2 million, greater than 1.3million, greater than 1.4 million, greater than 1.5 million, greaterthan 1.6 million, greater than 1.7 million, greater than 1.8 million orgreater than 1.9 million.

Ranges intermediate to the recited values are also intended to be partof this invention. For example, in the compositions of hyaluronanprovided herein, the average molecular weight of hyaluronan is between 1and 2 million, between 1 and 1.5 million, between 0.5 and 1 million,between 0.5 and 2 million, or between 0.9 and 1.4 million.

In some embodiments of the compositions of hyaluronan described herein,the majority of the hyaluronan present in the composition falls withinthe average molecular weight range described herein. Thus, for instance,in compositions with an average molecular weight of hyaluronan ofbetween 0.2 and 2 million, at least 95% of the hyaluronan present in thecomposition falls within the range of between 0.2 and 2 million. In someembodiments, at least 50% of the hyaluronan present in the compositionsprovided herein falls within the recited range of average molecularweight. In some embodiments, at least 60% of the hyaluronan present inthe compositions provided herein falls within the recited range ofaverage molecular weight. In some embodiments, at least 70% of thehyaluronan present in the compositions provided herein falls within therecited range of average molecular weight. In some embodiments, at least80% of the hyaluronan present in the compositions provided herein fallswithin the recited range of average molecular weight. In someembodiments, at least 90% of the hyaluronan present in the compositionsprovided herein falls within the recited range of average molecularweight. In some embodiments, at least 95% of the hyaluronan present inthe compositions provided herein falls within the recited range ofaverage molecular weight. In some embodiments, at least 98% of thehyaluronan present in the compositions provided herein falls within therecited range of average molecular weight. In some embodiments, at least99% of the hyaluronan present in the compositions provided herein fallswithin the recited range of average molecular weight. In someembodiments, at least 99.9% of the hyaluronan present in thecompositions provided herein falls within the recited range of averagemolecular weight.

II. Sources of Hyaluronan

The hyaluronan used in the compositions and methods described herein maybe obtained from any source. In general, hyaluronan has the samechemical structure, regardless of its origin (e.g., chicken or roostercomb, human tissue or bacterial cell wall). Hyaluronan can be obtained,for instance, from chicken or rooster comb, from bacterial cell wallsand from human tissue (umbilical cord, vitreous of the eye, synovialfluid from the joints, etc.). In some embodiments, the hyaluronan isisolated from chicken combs. In some embodiments, the hyaluronan isisolated from human tissue e.g., umbilical cord, vitreous of the eye,synovial fluid from the joints. In some embodiments, the hyaluronan isisolated from cell culture. In some embodiments, the hyaluronan isisolated from bacterial cell walls. The isolation of hyaluronan fromvarious sources is known to a person of ordinary skill in the art. Forinstance, the harvest and purification of hyaluronan from rooster combsis described in U.S. Pat. No. 4,141,973, while the harvest andpurification of hyaluronan from bacterial sources is described in U.S.Pat. No. 4,517,295. In some embodiments, the hyaluronan is purified andharvested to a solution with 0.15 M NaCl at a pH of 6-8. Generally, thehyaluronan obtained from the various sources will be free of proteins orglycosaminoglycans other than hyaluronan.

In some embodiments, the isolated hyaluronan is further purified toobtain hyaluronan with a desired average molecular weight range (e.g.,through column chromatography). Methods for purifying hyaluronan with adesired average molecular weight range are known to a person of ordinaryskill in the art.

In one aspect, the hyaluronan with high elasticity disclosed herein isunmodified hyaluronan. However, it should be appreciated that in someembodiments, the hyaluronan may be chemically modified. For instance,the hyaluronan may be chemically modified to increase the elasticity ofthe hyaluronan.

III. Sterilization of the Hyaluronan Compositions

In some embodiments, the HA compositions described herein and used inthe methods of the invention are sterile. A “sterile composition”, asused herein, refers to a composition that is safe to be administered toa subject, e.g., a human subject. Thus, a sterile composition will onlyhave a minimal number of agents that can cause unwanted side effectssuch as an unwanted tissue response, immune response, an inflammation oran infection.

Methods for sterilizing compositions of hyaluronan are known in the artand include, for example, heat or steam sterilization, e.g., byautoclaving. In some embodiments, the HA compositions of the inventionare sterilized by heating the compositions. In some embodiments, the HAcompositions of the invention are sterilized by including the HAcomposition in a syringe and autoclaving the HA containing syringe at131° C. for 2 minutes or 121° C. for 15 minutes followed by immediatecooling.

IV. Additional Components for the Hyaluronan Compositions

The HA compositions described herein may include additional componentsthat may stabilize the hyaluronan and/or make the composition moresuitable for administration to a subject. In some embodiments, the HAcompositions of the invention may include a buffer. Buffers are added inorder to allow for a stable pH. Suitable buffers for use in the presentinvention include phosphate buffers and bicarbonate buffers. In someembodiments, the buffer is a tris-phosphate buffer. In some embodiments,the buffer is present in a concentration of between 1 mM and 100 mM,between 2 mM and 50 mM, or between 5 mM and 20 mM. In some embodiments,the buffer concentration is less than 1 mM. In some embodiments, thebuffer concentration is more than 100 mM. In some embodiments, thebuffer concentration is 10 mM. It should be appreciated that the bufferconcentration is dependent on the nature of the buffer that is beingused. In some embodiments, the pH of the composition is between pH 7 andpH 9 or between pH 7.5 and pH 8.5. In some embodiments, the pH of thecomposition is 8.0. In some embodiments, the pH of the composition is7.5. In some embodiments, the pH of the composition is 8.5. If needed,acid (such as HCL) or base (such as NaOH) can be added to thecomposition to attain the desired pH.

For example, the HA compositions may include a buffer, e.g., aphysiologically compatible buffer, but do not include any additionalcomponents.

The HA compositions may also include a stabilizing excipient, such ascarboxylic acid or a salt thereof. In some embodiments, the compositionincludes a monocarboxylic acid and/or salt thereof. In some embodiments,the composition includes a gluconic acid and/or sodium gluconate. Insome embodiments, the composition includes a dicarboxylic acid and/or asalt thereof. In some embodiments, the composition includes a citricacid, succinic acid, malonic acid, maleic acid, tartaric acid and or asalt thereof. In some embodiments, the carboxylic acid is sodiumcitrate. In some embodiments, the composition includes a tricarboxylicaid (TCA) and/or a salt thereof. In some embodiments, the compositionincludes a nitrilotriacetic acid and/or sodium nitrilotriacetic acid. Insome embodiments, the composition includes a tetracarboxylic acid and/orsalt thereof. In some embodiments, the composition includes anethylenediaminetetracetic acid (EDTA) and/or sodium EDTA. In someembodiments, the composition includes a pentacarboxylic acid and/or asalt thereof. In some embodiments, the composition includes adiethylenetriaminepentaacetic acid (DTPA) and/or sodium DTPA. Suitablecarboxylic acids include, but are not limited to, citrate compounds,such as sodium citrate; tartrate compounds, succinate compounds, andEDTA. Kaushil et al. in Protein Science 1999 8: 222-233, and Busby etal. in the Journal of Biological Chemistry Volume 256, Number 23 pp12140-1210-12147 describe carboxylic acids and their uses. In someembodiments, the stabilizing excipient has a concentration of between 50to 600 mM, between 250 to 500 mM, or between 250 to 350 mM. In someembodiments, the concentration of the stabilizing excipient is 300 mM.In some embodiments, the concentration of the stabilizing excipient isless than 100 mM. In some embodiments, the concentration of thestabilizing excipient is more than 600 mM.

The HA compositions described herein may also include a sugar (e.g., adisaccharide sugar). Disaccharide sugars that can be added to thecomposition include, but are not limited to, sucrose, lactulose,lactose, maltose, trehalose, cellobiose, dextrose and dextran. In somecases, the sugar may be present at between 0.5 to 5% (wt/volume). Insome cases, the sugar may be present at between 1 to 2% (wt/volume). Inone embodiment, the sugar may be present at 1%. In some embodiments, thesugar may be present at less than 1% (wt/volume). In some embodiments,the sugar may be present at more than 5% (wt/volume). In one embodiment,the sugar may be sucrose or trehalose and is present at 1% (wt/volume).

In some embodiments, the HA compositions described herein may includesalts. Salts that can be used in the compositions include sodiumchloride and other physiological compatible salts. In some embodiments,the salt concentration present in the HA compositions described hereinis between about 10 mM and about 250 mM, between about 25 mM and about100 mM, between about 30 mM and about 70 mM, between about 45 mM andabout 150 mM, between about 125 mM and about 200 mM, between about 150mM and about 250 mM, or between about 190 mM and about 250 mM. In someembodiments, the salt concentration is 50 mM. In some embodiments, thesalt concentration is less than 10 mM. In some embodiments, the saltconcentration is more than 250 mM. In a specific embodiment, the saltconcentration is between about 50 mM and about 200 mM.

In some embodiments, the HA compositions of the invention may have anormal osmolarity, e.g., about 310 mOsm/L. In other embodiments, the HAcompositions of the invention may have an osmolarity that is below thenormal osmolarity, e.g., below 310 mOsm/L. For example, the HAcompositions of the invention may have osmolarity of between about 20mOsm/L and about 500 mOsm/L, e.g., between about 150 and about 310mOsm/L, between about 150 and about 200 mOsm/L, between about 160 andabout 220 mOsm/L, between about 180 and about 250 mOsm/L, between about200 and about 300 mOsm/L, between about 250 and about 310 mOsm/L,between about 290 and about 310 mOsm/L, between about 250 and about 290mOsm/L or between about 270 and about 300 mOsm/L. In some embodiments,the osmolarity of the HA compositions of the invention may be about 150mOsm/L, about 155 mOsm/L, about 160 mOsm/L, about 165 mOsm/L, about 170mOsm/L, about 175 mOsm/L, about 180 mOsm/L, about 185 mOsm/L, about 190mOsm/L, about 195 mOsm/L, about 200 mOsm/L, about 205 mOsm/L, about 210mOsm/L, about 215 mOsm/L, about 220 mOsm/L, about 225 mOsm/L, about 230mOsm/L, about 235 mOsm/L, about 240 mOsm/L, about 245 mOsm/L, about 250mOsm/L, about 255 mOsm/L, about 260 mOsm/L, about 265 mOsm/L, about 270mOsm/L, about 275 mOsm/L, about 280 mOsm/L, about 285 mOsm/L, about 290mOsm/L, about 295 mOsm/L, about 300 mOsm/L, about 305 mOsm/L or about310 mOsm/L. In one specific embodiment, the osmolarity of the HAcompositions of the invention is between about 100 mOsm/L and about 400mOsm/L, e.g., about 100 mOsm/L, about 110 mOsm/L, about 120 mOsm/L,about 130 mOsm/L, about 140 mOsm/L or about 150 mOsm/L.

In some embodiments, the HA compositions described herein include one ormore antioxidants. Antioxidants are substances capable of inhibitingoxidation by removing free radicals from solution. Antioxidants are wellknown to those of ordinary skill in the art and include materials suchas ascorbic acid, ascorbic acid derivatives (e.g., ascorbylpalmitate,ascorbylstearate, sodium ascorbate, or calcium ascorbate), butylatedhydroxy anisole, buylated hydroxy toluene, alkylgallate, sodiummeta-bisulfate, sodium bisulfate, sodium dithionite, sodiumthioglycollic acid, sodium formaldehyde sulfoxylate, tocopherol andderivatives thereof, (d-alpha tocopherol, d-alpha tocopherol acetate,d-alpha tocopherol succinate, beta tocopherol, delta tocopherol, gammatocopherol, and d-alpha tocopherol polyoxyethylene glycol 1000succinate) monothioglycerol and sodium sulfite. Such materials aretypically added in ranges from 0.01 to 2.0%.

In some embodiments, the HA compositions may include one or moreisotonicity agents. This term is used in the art interchangeably withiso-osmotic agent, and is known as a compound which can be added to acomposition to increase the osmotic pressure, such as an osmoticpressure of 0.9% sodium chloride solution, which is iso-osmotic withhuman extracellular fluids, such as plasma. Preferred isotonicity agentsthat can be used in the HA compositions include are sodium chloride,mannitol, sorbitol, lactose, dextrose and glycerol.

In some embodiments, the HA compositions of the invention may includeone or more preservatives. Suitable preservatives include but are notlimited to: chlorobutanol (0.3-0.9% w/v), parabens (0.01-5.0%),thimerosal (0.004-0.2%), benzyl alcohol (0.5-5%), phenol (0.1-1.0%), andthe like.

In some embodiments, the HA compositions may include components that aresuitable for ophthalmic use.

In some embodiments, the HA compositions may include one or morecomponents that minimize unwanted side-effects during injection of thecomposition.

V. Hyaluronan Compositions for Ophthalmic Uses

The present invention provides methods for alleviating pain anddiscomfort associated with a dry eye condition by administering to aneye of a subject compositions containing HA.

Any of a variety of carriers may be used in the HA compositions forophthalmic use, including water, mixtures of water and water-misciblesolvents, such as C1- to C7-alkanols, vegetable oils or mineral oilscomprising from 0.5 to 5% non-toxic water-soluble polymers, naturalproducts, such as gelatin, alginates, pectins, tragacanth, karaya gum,xanthan gum, carrageenin, agar and acacia, starch derivatives, such asstarch acetate and hydroxypropyl starch, and also other syntheticproducts, such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinylmethyl ether, polyethylene oxide, preferably cross-linked polyacrylicacid, such as neutral Carbopol, or mixtures of those polymers. Theconcentration of the carrier is, typically, from 1 to 100,000 times theconcentration of the active ingredient.

Additional ingredients that may be included in the HA compositions forophthalmic use include tonicity enhancers, preservatives, solubilizers,non-toxic excipients, demulcents, sequestering agents, pH adjustingagents, co-solvents and viscosity building agents.

For the adjustment of the pH, preferably to a physiological pH, buffersmay especially be useful. The pH of the hyaluronan compositions suitablefor administration to the eye should be maintained within the range of4.0 to 8.0, more preferably about 4.0 to 6.0, more preferably about 6.5to 7.8. Suitable buffers may be added, such as boric acid, sodiumborate, potassium citrate, citric acid, sodium bicarbonate,tris(hydroxymethyl)aminomethane (TRIS), and various mixed phosphatebuffers (including combinations of Na₂HPO₄, NaH₂PO₄ and KH₂PO₄) andmixtures thereof. Generally, buffers will be used in amounts rangingfrom about 0.05 to 2.5 percent by weight, and preferably, from 0.1 to1.5 percent.

Tonicity is adjusted, if needed, typically by tonicity enhancing agents.Such agents may, for example, be of an ionic and/or a non-ionic type.Examples of ionic tonicity enhancers include alkali metal or earth metalhalides, such as, for example, CaCl₂), KBr, KCl, LiCl, Nal, NaBr orNaCl, Na₂SO₄ or boric acid. Non-ionic tonicity enhancing agents include,for example, urea, glycerol, sorbitol, mannitol, propylene glycol, ordextrose. The osmolarity of hyaluronan compositions used in the methodsof the present invention may be adjusted with tonicity agents toapproximate the osmotic pressure of normal lachrymal fluids which isequivalent to a 0.9% solution of sodium chloride or a 2.5% solution ofglycerol. In some examples, an osmolarity of between about 100 mOsm/Land about 150 mOsm/L is preferred, e.g., 100 mOsm/L, 110 mOsm/L, 120mOsm/L, 120 mOsm/L, 130 mOsm/L, 140 mOsm/L or 150 mOsm/L. In otherexamples, an osmolality of between about 225 and 400 mOsm/L ispreferred, e.g., between about 280 and about 320 mOsm/L, or betweenabout 297 and about 318 mOsm/L. In some cases, the average osmolaritycan fluctuate between about 303.7 and about 306.7 mOsm/L.

In certain embodiments, the HA compositions for ophthalmic use mayadditionally comprise a preservative. A preservative may, typically, beselected from a quaternary ammonium compound such as benzalkoniumchloride (N-benzyl-N—(C8-C18 alkyl)-N,N-dimethylammonium chloride),benzoxonium chloride or the like. Examples of other suitablepreservatives include alkyl-mercury salts of thiosalicylic acid, suchas, for example, thiomersal; phenylmercuric nitrate, phenylmercuricacetate or phenylmercuric borate; sodium perborate; sodium chlorite;parabens, such as, for example, methylparaben or propylparaben;alcohols, such as, for example, chlorobutanol, benzyl alcohol or phenylethanol; guanidine derivatives, such as, for example, chlorohexidine orpolyhexamethylene biguanide; imidazolidinyl urea (Germall™) or sorbicacid. Preferred preservatives are quaternary ammonium compounds, inparticular benzalkonium chloride or its derivative such as Polyquad (seeU.S. Pat. No. 4,407,791), alkyl-mercury salts and parabens. Whereappropriate, a sufficient amount of preservative is added to theophthalmic composition to ensure protection against secondarycontaminations during use caused by bacteria and fungi.

In another embodiment, the HA compositions for ophthalmic use do notinclude a preservative. Such compositions would be useful for patientswho wear contact lenses, or those who use several topical ophthalmicdrops and/or those with an already compromised ocular surface (e.g., dryeye) wherein limiting exposure to a preservative may be more desirable.

The HA compositions for ophthalmic use may additionally require thepresence of a solubilizer, in particular if the active or the inactiveingredients tends to form a suspension or an emulsion. Solubilizerssuitable for the hyaluronan compositions used in the methods of theinvention include, for example, tyloxapol, fatty acid glycerolpolyethylene glycol esters, fatty acid polyethylene glycol esters,polyethylene glycols, glycerol ethers, a cyclodextrin (for examplealpha-, beta- or gamma-cyclodextrin, e.g. alkylated, hydroxyalkylated,carboxyalkylated or alkyloxycarbonyl-alkylated derivatives, or mono- ordiglycosyl-alpha-, beta- or gamma-cyclodextrin, mono- ordimaltosyl-alpha-, beta- or gamma-cyclodextrin or panosyl-cyclodextrin),polysorbate 20, polysorbate 80 or mixtures of these compounds. Apreferred solubilizer may be a reaction product of castor oil andethylene oxide, for example the commercial products Cremophor EL® orCremophor RH40®. Reaction products of castor oil and ethylene oxide haveproved to be particularly good solubilizers that are tolerated extremelywell by the eye. Another preferred solubilizer may be tyloxapol orcyclodextrin. The concentration used depends especially on theconcentration of the hyaluronan in the composition. The amount added istypically sufficient to solubilize the hyaluronan in the composition.For example, the concentration of the solubilizer is from 0.1 to 5000times the concentration of the hyaluronan in the composition.

The HA compositions for ophthalmic use may also comprise furthernon-toxic excipients, such as, for example, emulsifiers, wetting agentsor fillers, such as, for example, the polyethylene glycols designated200, 300, 400 and 600, or Carbowax designated 1000, 1500, 4000, 6000 and10000. The amount and type of excipient added is in accordance with theparticular requirements and is generally in the range of fromapproximately 0.0001 to approximately 90% by weight.

Other compounds may also be added to the formulations of the presentinvention to increase the viscosity of the carrier. Examples ofviscosity enhancing agents include, but are not limited to,polysaccharides, such as chondroitin sulfate and its salts, dextrans,various polymers of the cellulose family; vinyl polymers; and acrylicacid polymers.

In one embodiment, the hyaluronan compositions intended for ophthalmicuse, in addition to HA, may include additional ingredients that aretypically found in ophthalmic compositions. Examples of such componentsmay include other active ingredients, including, but not limited to,vasoconstrictors, antiallergenic agents, antiinfectives, steroids,anesthetics, anti-inflammatories, analgesics, dry eye agents (e.g.,secretagogues, mucomimetics, polymers, lipids, antioxidants), or beadministered in conjunction (simultaneously or sequentially) withcompositions comprising other active ingredients, including, but notlimited to, vasoconstrictors, antiallergenic agents, antiinfectives,steroids, anesthetics, anti-inflammatories, analgesics or dry eye agents(e.g., secretagogues, mucomimetics, polymers, lipids, antioxidants).

In some embodiments, the hyaluronan compositions of the inventions areadministered to an ocular surface of a subject, such as under the eyelid, e.g., under the upper or lower eye lid, of the subject or at thecornea-eyelid interface of the subject. In some embodiments, thehyaluronan compositions of the invention are not suitable for use, orare not used, as a viscosurgical tool or a device during ophalmicsurgery, i.e., are not suitable for injecting, or are not injected, intothe eye during ocular surgery.

In one example, the hyaluronan compositions of the invention areadministered to a subject immediately prior to rest or sleep.

VI. Hyaluronan Compositions for Skin Care/Dermatological Uses

The present invention also provides methods for alleviating pain anddiscomfort while minimizing at least one skin imperfection in a subjectin need thereof by administering compositions comprising HA. Such HAcompositions intended for skin care/dermatological use, in addition toHA, may include additional ingredients that are typically found in suchcompositions, e.g., compositions that are used as dermal fillers. Suchingredients may include, e.g., collagen, carnitine, Vitamin E, Vitamin Aand chondroitin sulfate.

In some embodiments, an HA composition for skin care/dermatological useis injected into the skin. Accordingly, it may be formulated in the formof a container filled with a HA composition, e.g., a pre-filled syringe.Any pre-filled syringes known to one of skill in the art may be used incombination with a composition of the invention. Pre-filled syringesthat may be used are described in, for example, in PCT PublicationsWO05032627, WO08094984, WO9945985, WO03077976, U.S. Pat. Nos. 6,792,743,5,607,400, 5,893,842, 7,081,107, 7,041,087, 5,989,227, 6,807,797,6,142,976, 5,899,889, US Patent Publications US20070161961A1,US20050075611A1, US20070092487A1, US20040267194A1 or US20060129108A1.Pre-filled syringes may be made of various materials. In one embodimenta pre-filled syringe is a glass syringe. In another embodiment apre-filled syringe is a plastic syringe. One of skill in the artunderstands that the nature and/or quality of the materials used formanufacturing the syringe may influence the stability of an HAcomposition stored in the syringe. In one embodiment, a pre-filledsyringe comprises a silicone-based lubricant. In one embodiment, apre-filled syringe comprises baked-on silicone. In another embodiment, apre-filled syringe is free from silicone-based lubricants. One of skillin the art also understands that small amounts of contaminating elementsleaching into the formulation from the syringe barrel, syringe tip cap,plunger or stopper may also influence the stability of the composition.For example, it is understood that tungsten introduced during themanufacturing process may adversely affect formulation stability. In oneembodiment, a pre-filled syringe may comprise tungsten at a level above500 ppb. In another embodiment, a pre-filled syringe is a low tungstensyringe. In another embodiment, a pre-filled syringe may comprisetungsten at a level between about 500 ppb and about 10 ppb, betweenabout 400 ppb and about 10 ppb, between about 300 ppb and about 10 ppb,between about 200 ppb and about 10 ppb, between about 100 ppb and about10 ppb, between about 50 ppb and about 10 ppb, between about 25 ppb andabout 10 ppb.

VII. Hyaluronan Compositions for Wound Healing

The present invention also provides methods for alleviating pain anddiscomfort while facilitating wound healing in a subject in needthereof. Such HA compositions intended for topical use, in addition toHA, may include additional ingredients that are typically found in suchcompositions. Such ingredients may include, e.g., 1,3-butylene glycol,glycerine, xanthan gum, sodium chondroitin sulfate, ethanol, methylp-hydroxybenzoate, polyoxyethylene-polyoxypropylene, decyltetradecylether, sodium citrate, sodium edetate and vitamins, e.g., Vitamin A,Vitamin B, Vitamin C, Vitamin D, Vitamin E and Vitamin K. Suchingredients may also include additional pharmaceutically activesubstances. In some cases, these additional pharmaceutically activesubstances do not comprise analgesics for treating pain. In other cases,these pharmaceutically active substances comprise antibiotics, e.g.,antibiotics that are typically used to treat skin infections and areintended for topical use.

The HA compositions for use in the methods disclosed herein alsoencompass a finished, packaged and labeled pharmaceutical product. Thisarticle of manufacture includes the appropriate unit dosage form in anappropriate vessel or container such as a glass vial, pre-filled syringeor other container that is hermetically sealed. In one embodiment, theunit dosage form is provided as a sterile particulate free HAcomposition that is suitable for parenteral administration, e.g.,intra-dermal or sub-dermal administration to a subject, e.g., the face,neck, arms, legs, or back, of a subject.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question, as well as how and how frequently toadminister the HA composition. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information.

VIII. Methods for Alleviating Pain and Discomfort Associated with DryEyes

Dry eye disease is an ocular disease affecting approximately 10-20% ofthe population. It may be associated with a pathological condition, suchas cataract or an autoimmune disorder, or, in the absence of apathological condition, may result under certain circumstances, such assuch as prolonged visual tasking, working on a computer, being in a dryenvironment, contact lens use or exposure to medications that result indrying of the eye surface.

In individuals suffering from dry eye, the reflex that results inblinking and the secretion of supportive tear substances is compromised.Signs and symptoms of dry eye include ocular dryness; decreased tearproduction, volume, and flow; abnormal tear composition; increased tearosmolarity; keratitis, conjunctival and corneal staining; redness;blurry visions; decreased tear film break-up time; increasedconjunctival redness; excess debris in tear film; ocular grittiness;ocular burning; foreign body sensation in the eye; excess tearing;photophobia; ocular stinging; refractive impairment; ocular sensitivity;ocular irritation and discomfort resulting from prolonged contact lenswear. The excess tearing response may seem counterintuitive, but it is anatural reflex response to the irritation and foreign body sensationcaused by the dry eye. Some individuals may also experience ocularitching due to a combination of ocular allergy and dry eye symptoms.

There are many possible variables that also can influence a person'ssymptoms of dry eye, including levels of circulating hormones, variousautoimmune diseases (e.g., Sjorgren's syndrome and systemic lupuserythematosus), ocular surgeries including PRK or LASIK, manymedications, environmental conditions, visual tasking such as computeruse, ocular fatigue, contact lens wear, and mechanical influences suchas corneal sensitivity, partial lid closure, surface irregularities(e.g., pterygium), and lid irregularities (e.g., ptosis,entropion/ectropion, pinguecula). Environments with low humidity, e.g.,those that cause dehydration, can exacerbate or cause dry eye symptoms,such as sitting in an aeroplane or a car with the defroster on or livingin a dry climate zone. In addition, visual tasking can also exacerbatesymptoms. Tasks that can greatly influence symptoms include watching TVor using a computer for long periods of time where the blink rate isdecreased.

Compositions containing HA have been previously used for treating dryeye conditions. HA-containing eye drops are commercially available andtypically contain HA at a concentration of less than 1%. Exemplarycommercially available eye drops containing HA include Opticalm eyedrops (0.2% HA); Aquify comfort drops (0.1% HA); Blink (0.15% HA);Hyal-drop (0.2% HA); Hycosan (0.1% HA); Oxyal (0.15% HA); and Vismed(0.18% and 0.8% HA).

Applicants have surprisingly discovered that highly concentrated HAcompositions that comprise HA at a concentration of greater than about30 mg/mL (or 3% HA), e.g., 40 mg/mL (or 4% HA) may be effectivelyadministered to an eye of a subject for treating a dry eye condition.Such highly concentrated HA compositions have not been previously usedfor treating dry eyes, as it was considered that these compositions weretoo viscous to be administered to an eye effectively.

Applicants have also surprisingly discovered that, once administered toan eye, concentrated HA compositions are surprisingly effective attreating pain and discomfort associated with a dry eye condition.Without wishing to be bound by a specific theory, it is believed thatpain associated with a dry eye condition may be modulated by TRPV1channels of nociceptors, and that HA molecules in highly concentrated HAcompositions interact with the TRPV1 channels present on ocular sensorynerves and corneal cells, thereby reducing the responsiveness of thenociceptors to noxious stimuli. Accordingly, in some embodiments, thepain and discomfort associated with a dry eye condition is modulated byreceptor potential vanilloid subtype 1 (TRPV1) channels. Moreover, thecharacteristic elastoviscous properties of the concentrated HA reducethe removal by normal tearing and blinking of the solution applied onthe eye surface, thereby prolonging the protective effects of the HAsolution.

The present invention provides a method for alleviating pain anddiscomfort associated with a dry eye condition in a subject in needthereof, comprising administering to an eye of the subject a compositioncomprising hyaluronan, wherein the hyaluronan is present in thecomposition at a concentration of greater than about 30 mg/mL; thehyaluronan has an average molecular weight of between about 1 and about2 million; and the hyaluronan is not cross-linked and/or issubstantially free of chemical modifications, thereby alleviating saidpain and discomfort in said subject.

In some cases, the HA compositions intended for ophthalmic use are inthe form a gel, an ointment, a liniment, a lotion or a cream. In certainaspects, this HA composition is not in the form of a lens or amicrosphere.

The HA compositions intended for ophthalmic use may be administered tothe ocular surface, such as under the eye lid e.g., under the upper orlower eye lid, or at the cornea-eyelid interface. In one aspect, the HAcompositions are not injected into the eye of the subject. Because thesubject's ability to see clearly may be compromised immediately afterthe administration of the HA compositions due to their high viscosity,these compositions may be administered to the eye of the subjectimmediately prior to rest or sleep.

One of ordinary skill in the art would be able to ascertain theappropriate administration schedule for the ophthalmic HA compositionsdescribed herein in order to achieve alleviation of pain and discomfortassociated with a dry eye condition. For example, the HA compositionsmay be administered daily for about 3 days, 4 day, 5 days, 6 days, 1week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, etc., or for anylength of time until alleviation of pain and discomfort associated witha dry eye condition is achieved. In some embodiments, such alleviationof pain and discomfort may be alleviated for long term, e.g., lastingfor about 12 hours, about 24 hours, about 1 day, about 3 days, about 5days, about 7 days, about 14 days or about 28 days.

The term “dry eye condition” includes, but is not limited to, a dry eyecondition associated with one or more symptoms selected from the groupconsisting of ocular dryness; decreased tear film break-up time;decreased tear production, volume, and flow; abnormal tear composition;increased tear osmolarity; keratitis; conjunctival and corneal staining;redness; blurry vision; increased conjunctival redness; excess debris intear film, ocular grittiness; ocular burning; foreign body sensation inthe eye; excess tearing; photophobia; ocular stinging; refractiveimpairment; ocular sensitivity; ocular irritation and discomfortresulting from prolonged contact lens wear.

The dry eye condition may also be associated with a condition selectedfrom the group consisting of an autoimmune disorder; an ocular surgery;ingestion of a medication; dry environmental conditions; prolongedcomputer use; ocular fatigue; contact lens wear, corneal sensitivity;partial lid closure; surface irregularities; eye lid irregularities; anda condition associated with neuropathic pain, such as cataract orretinal detachment. In one example, the dry eye condition may beassociated with an ocular surgery, such as photorefractive surgery,e.g., photorefractive keratectomy (PRK), cataract surgery, retinaldetachment surgery, laser-assisted in situ keratomileusis (LASIK), orany corneal surgical procedure involving damage to corneal sensorynerves.

IX. Methods for Alleviating Pain and Discomfort while Minimizing SkinImperfections

HA is the major component of the extracellular matrix (ECM) and ispresent in particularly large quantities in the soft connective tissues,such as the skin. In normal skin, HA is mainly synthesized by dermalfibroblasts and epidermal keratinocytes. With its residues bearing anegative charge, HA functions as a water pump for maintaining thehydration and elasticity of the skin. The HA has a main role incontrolling the distribution of food, hormones, vitamins and inorganicsalts of the connective tissue and in cleaning metabolic waste which mayinduce inflammatory reactions. With age, the amount of HA and its degreeof polymerization decreases, resulting in a decrease in the amount ofwater retained in the connective tissue. The skin is then subjected toan aging process which results in an increase of fibrosis and a decreasein the quality of elastic fibers.

HA has been widely used for cosmetic applications that include both theuse of HA compositions in the form of creams or gels for topicalapplications and the use of HA compositions for injecting into the skinas dermal fillers. The latter use involves injecting HA compositionsinto the top skin layers of the a subject, e.g., the face, neck, arms,legs, torso or chest, of a subject. This use results in the reduction ofwrinkles due to a mechanical filling effect of the cutaneous depressionresulting from the wrinkle, and due to a preventive effect against skinaging and degradation of the ECM that is essential to maintaining themechanical properties of the skin elasticity and firmness.

Although injection of dermal fillers for soft tissue augmentation is aminimally invasive dermatological procedure, patients often expressconcern about pain associated with such a procedure. Topical anestheticcreams are often used to alleviate pain during these procedures, andanesthetics are also included in the HA compositions used for injection.

The present invention obviates the need for an anesthetic use duringthese procedures. Specifically, the present invention provides methodsfor alleviating pain and discomfort associated with a dermatologicalprocedure while minimizing at least one skin imperfection in a subjectin need thereof by administering to the subject highly concentrated HAcompositions. As explained above, it was surprisingly discovered thatthe highly concentrated HA compositions described herein aresurprisingly effective at treating pain and discomfort associated with adermatological procedure that may comprise, e.g., an injection into theskin of a subject. Without wishing to be bound by a specific theory, itis believed that pain associated with injecting HA compositions into theskin may be modulated by TRPV1 channels of nociceptors, and that HAmolecules in highly concentrated HA compositions interact with the TRPV1channels present in sensory nerves and epithelium and connective tissuecells of the skin and subcutaneous tissue, thereby reducing theresponsiveness of the nociceptors to noxious stimuli.

In some embodiments, the HA compositions for cosmetic use may beadministered to a subject in need thereof via an injection, e.g.,subcutaneous or intradermal injection, using an injection device, suchas a needle, a trocar, a cannula or a perfusion device. The injectiondevice suitable for injecting the HA compositions of the invention mayhave a nominal diameter of 2.11 mm or greater (corresponding to 14Gneedle, or a needle gauge of 14 of greater). In some embodiments, the HAcompositions of the invention may be too viscous for administrationusing smaller needles, e.g., needles having a nominal diameter of lessthan 2.11 mm. In other embodiments, the HA compositions of the inventionmay allow administration using smaller injection devices having anominal diameter of less than 2.11 mm.

For example, a device suitable for injecting the HA compositions of theinvention, such as a syringe, may have a nominal diameter of about 0.31mm, 0.34 mm, 0.36 mm, 0.41 mm, 0.474 mm, 0.46 mm, 0.49 mm, 0.515 mm,0.51 mm, 0.54 mm, 0.57 mm, 0.59 mm, 0.642 mm, 0.64 mm, 0.67 mm, 0.718mm, 0.72 mm, 0.77 mm, 0.82 mm, 0.87 mm, 0.91 mm, about 0.99 mm, about1.07 mm, about 1.17 mm, about 1.27 mm, about 1.42 mm, about 1.47 mm,about 1.57 mm, about 1.65 mm, about 1.73 mm, about 1.83 mm, about 1.98mm, about 2.11 mm, about 2.26 mm, about 2.41 mm, about 2.54 mm or about2.77 mm, corresponding, respectively, to gauge of 30, 29, 28, 27, 26 s,26, 25.5, 25 s, 25, 24.5, 24, 23.5, 23 s, 23, 22.5, 22 s, 22, 21.5, 21,20.5, 20, 19.5, 19, 18.5, 18, 17.5, 17, 16.5, 16, 15.5, 15, 14.5, 14,13.5, 13, 12.5 or 12 (or 30 G, 29 G, 28 G, 27 G, 26 sG, 26 G, 25.5 G, 25sG, 25 G, 24.5 G, 24 G, 23.5 G, 23 sG, 23 G, 22.5 G, 22 sG, 22 G, 21.5G, 21 G, 20.5 G, 20 G, 19.5 G, 19 G, 18.5 G, 18 G, 17.5 G, 17 G, 16.5 G,16 G, 15.5 G, 15 G, 14.5 G, 14 G, 13.5 G, 13 G, 12.5 G or 12 G needles).In one embodiment, the HA compositions of the invention may beadministered using an 18G syringe needle having a nominal diameter ofabout 1.27 mm. In some embodiments, the HA compositions of the inventionmay be too viscous for administration using smaller needles, e.g.,needles having a nominal diameter of less than 1.27 mm.

In some embodiments, the methods of the present invention may alsocomprise administering to a subject highly concentrated HA compositionsin the form of an injectable implant, in particular, a dermal implant.

The compositions according to the invention are particularly intendedfor use in humans or animals in reconstructive or plastic surgery orcosmetic dermatology for filling wrinkles, fine lines, skin depressions,and scars, including the filling of skin depressions caused bylipodystrophy or lipoatrophy. The composition may be an implant asdefined above.

X. Methods for Alleviating Pain and Discomfort while Facilitating WoundHealing

Skin provides a mechanical barrier to the external environment and actsto prevent the ingress of infectious agents. Once injured, the tissuesbeneath are exposed to infection; therefore, rapid and effective healingis of crucial significance to reconstruct a barrier function. Skin woundhealing in adults is a complex process, and includes multiple stages,such as inflammation, granulation tissue formation, reepithelization andremodeling. HA likely plays a multifaceted role in mediation of thesecellular and matrix events. Often, topical anesthetics are administeredto wounds in order to alleviate pain associated with these wounds.

The present invention provides methods for effectively treating skinwounds while simultaneously providing pain relief and obviating the needfor a topical anesthetic use. Specifically, the present inventionprovides methods for alleviating pain and discomfort while facilitatingwound healing, by administering to a subject in need thereof highlyconcentrated HA compositions. As explained above, it was surprisinglyfound that the highly concentrated HA compositions described herein aresurprisingly effective at treating pain and discomfort associated withwound healing. Without wishing to be bound by a specific theory, it isbelieved that pain associated with injecting HA compositions into theskin may be modulated by TRPV1 channels of nociceptors, and that HAmolecules in highly concentrated HA compositions interact with the TRPV1channels present in the sensory nerves innervating the wounded area andin connective tissue cells of this area, thereby reducing theresponsiveness of the nociceptors to noxious stimuli.

In accordance with the methods of the invention, the highly concentratedHA compositions intended for topical administration to skin wounds maybe administered topically on the surface of a wound or a scar on theskin. The compositions may be in the form of aqueous gels orpolyalcohols containing thickening polymers such as cellulosederivatives or acrylic polymers, together with other excipients inconventional use, such as preservatives, perfumes and the like.

A treatment, preventive or alleviating effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status or a pathological condition, e.g., pain and discomfortassociated with a dry eye condition, a skin care/dermatologicalprocedure or a healing wound. A treatment, or alleviating or preventiveeffect is also evident by a failure to worsen or to develop symptomswhere they would otherwise be anticipated. As an example, a favorablechange of at least 10% in a measurable parameter of disease orcondition, e.g., pain and discomfort, and, preferably at least 20%, 30%,40%, 50% or more can be indicative of effective treatment. The term“prevent” or “preventing”, as used herein, comprises, e.g., preventionof re-occurrence of pain and discomfort in a subject who has previouslyexperienced the pain.

In some embodiments, the subject is a human, a mammal, e.g., a domesticanimal (such as a cat or a dog), a farm animal (such as a cow, a sheep,a horse, a donkey), or a rodent (such as a guinea pig, a mouse or arat). In a specific embodiment, the subject is a human. In anotherspecific embodiment, the subject is a dog.

As used herein, the term “reducing at least one symptom” comprisesdiminishing, ameliorating or eliminating at least one symptom associatedwith pain and discomfort. This term also comprises reducing the extentof the activation of ion channels, such as TRPV1 channels, that areinvolved in the process of pain transduction in neurons, uponadministration of an HA composition of the invention. Activation of suchchannels may be measured, e.g., by measuring the change in intracellularCa′ in neurons after a nociceptive impulse, or by measuring whole-cellcurrents in neurons, upon administering an HA composition of theinvention. Furthermore, the term “reducing at least one symptom” alsocomprises diminishing nociceptive firing of neurons upon administrationof an HA composition of the invention.

In some embodiments, the methods of reducing pain and discomfortassociated with dry eye condition, a skin care/dermatological procedureor a healing wound, comprise administering to a subject in need thereofa therapeutically effective amount of a composition of the invention.The term “therapeutically effective amount”, as used herein, is intendedto include an amount of an HA composition of the invention that, whenadministered to a subject in need thereof, is sufficient to treat,prevent, reduce or alleviate pain and discomfort. One of ordinary skillin the art, e.g., a physician, would be able to easily ascertain theamount of HA composition that would be therapeutically effective. Ingeneral, a therapeutically effective amount of the composition isbetween about 0.1 to about 500 mg, e.g., about 0.1 mg, about 1 mg, about5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg,about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about460 mg, about 470 mg, about 480 mg, about 490 mg, or about 500 mg.

A variety of treatment regimens are encompassed by the methods disclosedherein. For instance, a subject may receive a first dose of the HAcompositions disclosed herein followed by additional doses. In someembodiments, a first dose is administered followed by a second dose at aspecific interval. In some embodiments, the second dose is administeredabout 30 days, about 60 days, about 90 days, about 120 days, about 150days, about 180 days, about 210 days, about 240 days, about 270 days,about 300 days, about 330 days, or about 360 days after the first dose.It should be appreciated that the dose regime may be adjusted based onthe reduction in pain and discomfort experienced by the subject. In someembodiments, the subject will receive a dose every month, every twomonths, every three months, every four months, every five months, everysix months, every seven months, every eight months, every nine months,every ten months, every eleven months, or every twelve months.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein by reference.

EXAMPLES

Unless provided otherwise, the hyaluronan (HA) used in the compositionsdescribed herein is obtained from animal, human or bacterial sources.Unless provided otherwise, the compositions used herein are inphysiological buffers.

Example 1. Reduction of Impulse Activity in Corneal Sensory Nerve Fibersby Topical Application of 4% Sodium Hyaluronan on the Cornea

Excitation of nociceptors is the first step in the production of painsensations. For acute pain, the magnitude of pain sensation correlateswell with the firing frequency of nociceptive fibers and with the numberof fibers recruited by the stimulus (Acosta et al., 2001, J. Physiol.534(2), 511-525). Also, sustained pain after peripheral injury isassociated with ongoing activity in nociceptors (Belmonte et al., 2004,Ocular Surface, 2, 248-253). Augmented sensory nerve impulse activity incorneal nerves is the first step in the generation of ocular discomfortand pain sensations. In the ocular surface, noxious stimuli are detectedby polymodal nociceptor and mechano-nociceptor innervating the corneaand conjunctiva (Belmonte & Giraldez, 1981, J. Physiol., 437, 709-725;Gallar et al., 1993, J. Physiol., 468, 609-622). When inflammationoccurs, polymodal nociceptors become sensitized and fire continuously,evoking sustained pain sensations. On the other hand, activity incorneal cold thermoreceptors has been associated with ocular surfaceevaporation and possibly also contributes to conscious sensations ofocular surface dryness (Gallar et al., 1993, J. Physiol., 468, 609-622;Parra et al., 2010, Nature Medicine, 16, 1396-1399; Kovacs et al., 2016,Pain, 157, 399-417).

The objective of the experiments described below was to test the abilityof hyaluronan at a concentration of 4% (4% HA) with an average molecularweight of 1-2 million to modify the spontaneous and stimulus-evokednerve impulse discharges in sensory receptor fibers(mechano-nociceptors, polymodal nociceptors, cold thermoreceptors)innervating the cornea of guinea pigs.

An effective means of producing a chemical stimulation of cornealpolymodal nociceptors is application of 98.5% CO₂, due to acidificationelicited by local formation of carbonic acid (Chen et al., 1995, Eur. J.Neurosci. 7, 1154-1163). It has been proven that application of asimilar pulse of CO₂ to the human cornea evokes in all instances animmediate and sharp sensation of pain (Chen et al., 1995, Eur. J.Neurosci. 7, 1154-1163; Acosta et al., 2001, J. Physiol. 534(2),511-525). Because protons are formed rapidly in the surroundings ofnerve terminals, the stimulating effect of H⁺ is presumably direct andoccurs through activation of a proton-gated ionic channel thatdepolarizes the nerve, possibly the ‘capsaicin channel’ TRPV1 and/orASIC channels. Moreover, a decrease of the intracellular pH inepithelial cells of the cornea subjected to CO₂ stimulation may alsolead to formation and release of inflammatory mediators (for instance,prostaglandins), which may sensitize corneal polymodal nociceptors,causing background discharge and enhanced impulse responses to directacidic stimulation.

In the case of corneal cold thermoreceptors, these fibers exhibit abackground, regular impulse activity at the basal corneal temperature of34° C. and increase their firing frequency with very small temperaturereductions, e.g., those occurring during tear evaporation (Belmonte etal., 2015, Curr. Ophthalmol. Rep. 3, 111-121).

Methods

Dunkin Hartley guinea pigs of both sexes weighing 200-300 g were used inthe experiments. The study was carried out in accordance with the NIHGuide for the Care and Use of Laboratory Animals and the European UnionDirective (2010/63/EU) and the Spanish regulations on the protection ofanimals used for research, and followed a protocol approved andsupervised by the Ethics Committees of the University Miguel Hernandez.

Electrophysiological Recordings

The impulse activity of different functional types of peripheral cornealreceptors was recorded. For this purpose, animals were euthanized withan intraperitoneal injection of 100 mg/kg sodium pentobarbitone, andboth eyes were immediately enucleated together with the bulbar andtarsal conjunctiva and the optic and ciliary nerves, and placed in coldsaline (4° C.).

Connective tissue and extraocular muscles were carefully removed fromthe excised eyeball to expose the back of the eye with the ciliarynerves around the optic nerve. The eye was then placed in a doublechamber specially designed to keep the anterior segment of the eye withthe conjunctiva separated from the back pole and the ciliary nerves. Inthe front part of the chamber, the conjunctiva was pinned to theseparating wall in order to isolate both compartments, which wereperfused separately. The anterior compartment was perfused with warm(34° C.) saline, dropping continuously over the upper corneoscleralborder. In the rear compartment of the chamber filled with warmedmineral oil, nerve filaments were teased apart from the ciliary nervesand placed on an Ag—AgCl electrode for monopolar recording of singleunit impulse activity using conventional electrophysiological equipment.Electrical signals were recorded with respect to an Ag/AgCl pellet inthe posterior compartment. Electrical signals were transferred to a PCwith a CED interface and analyzed with the appropriate software.Spontaneous activity of the selected unit was recorded for 1 minutebefore any intended stimulation. Mechanical threshold was determinedthereafter using calibrated von Frey hairs (range 0.25-4.00 mN).Receptive fields of corneal afferent fibers were localized usingmechanical stimulation with a fine paint brush and mapped afterwardsusing a suprathreshold von Frey hair. For chemical stimulation, a gasjet containing 98.5% CO₂ was applied on the corneal receptive fieldduring 30 seconds. Thermal stimulation was performed by cooling (down to20° C.) the perfusion solution by means of a custom-made Peltier device.

Data Analysis

In single fiber recordings of polymodal fibers, impulse firing ofindividual units discriminated accordingly to their stimulus modality,amplitude and shape was analyzed. Ongoing impulse activity was expressedas mean impulse frequency (in impulses/s) measured during 30 seconds atthe beginning of the recording and during the interstimulus periods.Responses to CO₂ were quantified measuring the following parameters:latency: time delay between onset of the CO₂ pulse and the first impulsegiven by the unit; mean discharge rate: mean number of impulses persecond (imp/s) throughout the CO₂ pulse; postdischarge: mean firingfrequency (imp/s) during 30 seconds immediately after the CO₂ pulse.

In cold nerve fiber recordings, the following additional firing patternparameters were calculated: mean firing frequency: average number ofimpulses recorded per second (impulses/s); cooling threshold:temperature during the cooling ramp at which a 25% increase of the meannerve impulse frequency at basal temperature was obtained; peakresponse: maximal impulse/s value of the impulse frequency during thecooling ramp; temperature at the peak response: temperature value (° C.)at which peak frequency was reached.

Experimental Protocol

The following experimental sequence was followed:

Identification of Polymodal Nociceptive Fibers

A filament containing fibers innervating the cornea and responding tomechanical stimulation with a wet, fine brush applied to the cornealsurface was identified. After splitting of the nerve filament,localization of a single corneal nociceptive unit was made by mechanicalstimulation. For further characterization (determination ofpolymodality) of each isolated single unit, the following routine wasperformed:

1. mapping of the receptive field borders and determination ofmechanical threshold with the von Frey filaments;

2. measuring a response to a 30 second pulse of 98.5% CO₂. If noresponse was detected, a new fiber was investigated.

Effect of 4% HA on Polymodal Nociceptors

A 4% HA solution was applied onto the corneal surface. The cornearegularly received a drop of saline to maintain humidity. After 4% HAinstillation, a 30-second CO₂ pulse was applied onto the corneal surfaceat successive times (5, 20, 35, 50, 65 and 80 min after 4% HAapplication). The cornea was then washed continuously for 5 minutes withsaline and the response to CO₂ measured 15 minutes and 20 minutes afterwashing. Electrical threshold was measured at the end of the protocol toconfirm responsiveness of the fiber. The presence of spontaneousactivity throughout the complete experimental procedure and themagnitude of the impulse response to the CO₂ stimuli were measured.

Identification of Cold Thermosensitive Fibers

A filament containing cold thermosensitive fibers innervating the corneawas identified by applying a drop of cold saline on the cornea. Aftersplitting of the fiber, localization of the filament containing a singlecorneal cold unit was made using an ice-cooled metal bar (tip diameter 1mm diameter) applied onto the cornea to map the receptive field.

Ongoing impulse activity exhibited by a single cold-sensitive nerveterminal at 34° C. was recorded. Cooling ramps from 34° C. to 15° C.were performed by changing the temperature of the receptive field with aPeltier device.

Effect of 4% HA on Cold Thermoreceptor Nerve Activity

A 4% HA was applied onto the ocular surface exposed in the frontcompartment of the chamber, and the effect on the cold thermoreceptorongoing activity at 34° C. and on the response to cooling ramps wasexplored 5, 20, 35, 50, 65 and 80 minutes after HA application, as wellas 15 and 20 minutes after washing the cornea.

The mean ongoing activity at the resting temperature of 34° C., thethermal threshold and the increase in nerve impulse activity during thecooling ramps were determined.

Results

In the intact cornea, sensory afferents identified as polymodalnociceptor fibers presented corneal receptive fields usually extendingup to 1 mm into the adjacent sclera. The polymodal nociceptor fiberswere silent at rest, responding to mechanical stimulation and also to a98.5% CO₂.

The effects of 4% HA on the ongoing activity and the firing response toCO₂ were measured in 2 single polymodal and 2 cold sensitive units. Theeffects of CO₂ were analyzed before and after application of 4% HA onthe corneal surface. As shown in Table 1 below, polymodal nociceptorswere silent at rest, with no ongoing activity during a 2-minuterecording period. They responded in about 2.5 seconds to a 30 second CO₂pulse with a mean frequency of the impulse discharge of about 6 impulsesper second. During the first 35 minutes after applying 4% HA onto thecornea, the response to CO₂ appeared markedly decreased and ceasedafterwards. Washing after 80 minutes of 4% HA treatment graduallyrecovered the response to CO₂.

TABLE 1 COLD THERMORECEPTORS POLYMODAL NOCICEPTORS Cooling Ongoing CO₂Post- Spontaneous ramp Peak Temp. Activity Latency response dischargeactivity Threshold response frequency at peak CONTROL 0  2.4 ± 3.3 5.8 ±4.1 1.3 ± 0.8 0.3 17.8 3.3 5.0 17.4 4% HA  5 min 0 0 0 0 0 16.0 0.5 3.015.7 20 min 0 9.6 ± 0  0.03 ± 0.04 0.02 ± 0.02 0 18.7 1.2 4.0 15.7 35min 0 23.9 ± 7.2 0.3 ± 0.2  0.6 ± 0.02 0 20.7 0.4 2.0 17.8 50 min 0 0 00 0.8 16.9 1.2 3.0 16.6 65 min 0 0 0 0 0.2 n.r. n.r. n.r. n.r. 80 min 00 0 0 0.3 15.8 0.5 1.0 15.8 WASH 15 min 0  8.4 ± 6.4 0.4 ± 0.1 0.8 ± 0.20.15 18.2 1.1 4.0 18.0 20 min 0 14.8 ± 5.8 4.5 ± 3.2 3.1 ± 1.9 0.0 21.01.5 8.0 17.3 Ongoing activity, CO2 responses and postdischarges valuesof corneal polymodal nociceptors are expressed in impulses per second;latency in seconds. For cold thermoreceptors, spontaneous activity,cooling ramp responses and peak frequency values were expressed inimpulses per second. Threshold and temperature at the peak frequency, in° C; n.r.: impulse activity, absent.

The impulse activity in cold thermoreceptor fibers, which representabout 10% of all corneal sensory afferents, was also recorded. Weconfirmed their regular ongoing impulse activity at 34° C. in intacteyes and the marked frequency increase caused by 15° C. cooling pulses,characteristic of ocular cold thermoreceptors (Gallar et al., 1993, J.Physiol., 468, 609-622; Parra et al., 2010, Nature Medicine, 16,1396-1399).

As shown in Table 1, exposure of the cornea for 4 hours to 4% HAdecreased the background ongoing activity of cold thermoreceptors at 34°C. No clear changes in cold threshold were noticed but, as also shown inTable 1, the response to cold ramps was markedly decreased and onlypartially recovered 20 minutes after washing, while the peak frequencyvalue evoked by the cold ramp returned to a normal value during thistime.

Altogether, these results indicate that 4% HA reduces the abnormalimpulse activity in polymodal nociceptor fibers resulting from acidicstimulation of cornea. This is possibly the result of an interaction ofHA molecules with the TRPV1 channels of sensitized nociceptive nerveterminals (Caires et al., 2015, Nat. Comm. 6, 8095), evidencing thathigh concentrations of HA are very effective at eliciting this effect.Also, activity in cold thermoreceptors was diminished, possibly due to amore effective shielding of the corneal surface than HA solutions oflower concentrations.

Example 2. Force Requirements for Ejection of HA Through Various NeedleSizes

The pressure required to eject a 4% HA composition from a 3 mL syringewith needles of different diameter (30-18 G) has been measured and isshown in FIG. 1 . Force was exerted by one plate of a two-plate balance,acting perpendicularly on the embolus of the syringe. Weights ofincreasing magnitude were added to the contralateral plate. Asdemonstrated by the results shown in FIG. 1 , the HA compositions of theinvention can be administered to subjects using needles with diametersof 30-18G.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

What is claimed is:
 1. A method for alleviating pain and discomfort associated with minimizing at least one skin imperfection in a subject in need thereof, the method comprising administering to said subject a composition comprising hyaluronan, wherein: the hyaluronan is present in said composition at a concentration of greater than about 30 mg/mL; the hyaluronan has an average molecular weight of between about 1 and about 2 million; the hyaluronan is not cross-linked and/or is free of chemical modifications; and wherein the composition is free of a local anesthetic, thereby alleviating said pain and minimizing said at least one skin imperfection.
 2. The method of claim 1, wherein hyaluronan is present in said composition at a concentration of about 40 mg/mL to about 60 mg/mL.
 3. The method of claim 1, wherein hyaluronan is present in said composition at a concentration of about 40 mg/mL.
 4. The method of claim 1, wherein the composition further comprises a buffer.
 5. The method of claim 4, wherein the buffer is phosphate buffered saline (PBS).
 6. The method of claim 1, wherein the composition has an elasticity of at least about 200 Pascal when measured at a frequency of 0.5 Hz.
 7. The method of claim 1, wherein the composition has an elasticity of at least about 1,000 Pascal when measured at a frequency of 0.5 Hz.
 8. The method of claim 1, wherein the composition has an elasticity of at least about 2,000 Pascal when measured at a frequency of 0.5 Hz.
 9. The method of claim 1, wherein the composition has an elasticity of at least about 4,000 Pascal when measured at a frequency of 0.5 Hz.
 10. The method of claim 1, wherein the composition is sterile.
 11. The method of claim 1, wherein said composition is administered by an injection into the skin of said subject.
 12. The method of claim 11, wherein said composition is injected into a region of the subject selected from the group consisting of the face, the neck, the arms, the legs, the torso and the chest of the subject.
 13. The method of claim 12, wherein said composition is injected into the region selected from the group consisting of nasolabial region, upper lip region, forehead, eye region and cheek region.
 14. The method of claim 11, wherein the composition is administered by an injection using a pre-filled syringe.
 15. The method of claim 14, wherein the pre-filled syringe is a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-mL pre-filled syringe.
 16. The method of claim 15, wherein the pre-filled syringe is sterilized. 